Project Summary ? Maintaining stable replication forks in spite of constant bombardment of DNA damage that can cause deleterious double strand breaks (DSBs) is an inherent genomic protective function during the most vulnerable period of DNA replication. Defects at various stages of replication fork remodeling are linked to several genetic diseases with increased susceptibilities to a multitude of endogenous or exogenous agents leading to cancer, intellectual disabilities linked to aneuploidy, and infertility. Two recently identified proteins, MCM8 and MCM9, have been associated with both mitotic and meiotic replication/recombination intermediates in mammals. Results from knockout mice, genetic mutations and phenotypes in humans, and functional cellular studies have only scratched the surface of our understanding, as their specific roles or activities have not been delineated. MCM8 and 9 are members of a broader AAA+ family of proteins and conserved within the MCM family that include ATP hydrolysis, DNA binding, and specific motifs that contribute to DNA unwinding. We hypothesize that the heterohexameric MCM8/9 complex aids in stalled replication fork remodeling for high fidelity replication restart or alternatively direct downstream recombination. Deficiencies due to mutations in MCM8 or MCM9 genes will disrupt the DNA repair process, induce cell cycle checkpoint activation, decrease recombination propensity, and increase cell death. To test this hypothesis, we will determine the ability of MCM8/9 to remodel stalled replication forks through epistatic relationships with known fork stabilizing, reversal and, restart components and directly measure fork outcomes after stalling. We will also characterize the intrinsic enzymatic abilities of MCM8 and 9 as a DNA helicase both in vitro and in vivo. The proposed research program is highly integrated using advanced cell biological, biochemical, and enzymological approaches to better understand MCM8/9 functions during repair and will be performed by several excellent undergraduate and graduate student researchers providing a significant health science training opportunity at a primarily undergraduate university. Results from this proposal will provide a clearer understanding of the specificity and activity of MCM8/9 to remodel stalled replications forks within the context of many other known DNA repair factors. Conclusions from these studies will be influential in describing molecular events that contribute to faithful genomic restoration processes and provide insight into consequences of patient mutations leading to cancer and infertility.